Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid

Abstract

BackgroundTriple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells.MethodsPrimary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed.ResultsThe C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs.ConclusionTKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.

Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as an alternative to human red blood cells for transfusion

“…The ABO type of this commercial serum was not provided; however, we presumed that AB-type hRBCs would be ABO-incompatible with this serum. Meanwhile, WT pig cells have been shown to activate complement via the alternate pathway ( 21 , 31 ). Accordingly, under Ca ++ -depleted conditions, which allow only alternative pathway activation, a significant increase in C3 deposition was found only on WT pRBCs and not on genetically modified pRBCs, suggesting that the surface of these genetically modified pRBCs is not prone to the activation of the human complement system, similar to hRBCs.…”

“…Two million RBCs in single-cell suspensions were incubated in 100 µL of 0, 10, 20, and 30% human C7-depleted serum diluted in Ca ++ - and Mg ++ -enriched gelatin veronal buffer (GVB ++ , for total complement activation) or Mg ++ -EGTA-GVB (for alternative pathway complement activation) at 37°C for 30 min and then stained with fluorescein-conjugated goat IgG fraction to human complement C3 (MP Biomedicals, Solon, OH, USA) ( 21 , 29 – 31 ). C7-depleted serum allows complement activation of RBCs but prevents complement-mediated lysis of RBCs during the assays.…”

“…In our previous study ( 20 ), anti-Gal and anti-Neu5Gc IgG and IgM antibodies were detected in most healthy Koreans, supporting the necessity of GT KO and CMAH KO gene modifications in donor pigs. Incidentally, altering sialic acid composition by CMAH KO gene modification does not hamper factor H-mediated complement inhibition in porcine cells incubated in human serum ( 21 ). When incubated in human platelet-rich plasma, TKO porcine cells showed delayed platelet aggregation compared with that in wild-type (WT) or GT KO/ CMAH KO porcine cells ( 21 ).…”

“…Incidentally, altering sialic acid composition by CMAH KO gene modification does not hamper factor H-mediated complement inhibition in porcine cells incubated in human serum ( 21 ). When incubated in human platelet-rich plasma, TKO porcine cells showed delayed platelet aggregation compared with that in wild-type (WT) or GT KO/ CMAH KO porcine cells ( 21 ). These results suggest that TKO genetic modification in donor pigs is a baseline platform for developing alternatives to hRBCs.…”